Sulfur-containing compound, method of preparation and pharmaceutical uses thereof

ABSTRACT

The invention relates to a sulfur-containing compound and the preparation thereof. The invention also relates to the uses of the sulfur-containing compound in inhibiting inducible nitric oxide synthase and/or cyclooxygenase-2 and in treating the diseases associated with inducible nitric oxide synthase and/or cyclooxygenase-2. This invention also describes a series of chemical analogues of the said sulfur-containing compound and the preparation of these compounds.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a novel sulfur-containing compound. Saidsulfur-containing compound has ability to inhibit the function ofinducible nitric oxide synthase (iNOS) and/or cyclooxygenase-2 (COX-2).

2. Description of the Related Art

With the progression of civilization, we human beings not only havelongevity, but also emphasize the quality of our daily lives. However, aspecific and effective drug is still absent for many diseases nowadays,such as cancer, chronic pain and atherosclerosis.

Inflammation has been proven to play an important role in the occurrenceof several diseases in many studies. The occurrence of theinflammation-related diseases is highly associated with chronic andlong-terminflammation induced by free radicals, pollution, food, ages,and pressure.

Atherosclerosis leads to remold a blood vessel and further causes thereduction of the inside diameter of the vessel. Therefore, it is animportant risk factor of one of the leading causes of death, acute andlethal cardiovascular diseases, such as myocardial infarction, strokeand peripheral vascular diseases (Libby, Am J Clin Nutr 83:456 S-460S,2006). Atherosclerosis is proven to be a chronic inflammatorycardiovascular disease (Ross, N Engl J Med 340: 115-126, 1999). Whenintima cells of the blood vessel are pressed or injured, monocytes areinduced to differentiate into macrophages and accumulate abundantlyaround the injured tissue. Through a series of inflammatory reactions,smooth muscle cells of the blood vessel proliferate and inflammatorycells accumulate, and such reactions damage the blood flow and lead tocardiovascular diseases finally (Lucas and Greaves, Exp Rev Mol Med3:1-18, 2001; Gordon, Bioessays 17:977-986, 1995). In animal modelstudies, the inflammatory critical factors of inducible nitric oxidesynthase and cyclooxygenase-2 are shown to play an important role inatherosclerosis (Cipollone, Lupus 14:756-759, 2005; Boyle, Curr VascPharmacol 3:63-68, 2005). Furthermore, bulk of inducible nitric oxidesynthase and cyclooxygenase-2 is expressed in the human atherosclerosistissue that comprises macrophages and proliferated smooth muscle cells(Baker et al, Arterioscler Thromb Vasc Biol 19:646-655, 1999; Buttery etal, Lab Invest 75:77-85, 1996). Presently, inducible nitric oxidesynthase and cyclooxygenase-2 inhibitors are proven to significantlyprevent the occurrence of atherosclerosis (Burleigh et al, Circulation105:1816-23, 2002; Hayashi et al, Atherosclerosis 187:316-324, 2006;Osiecki, Altern Med Rev. 9: 32-53, 2004).

According to the definition made by International Association for theStudy of Pain (IASP), pain is an unpleasant sensory and emotionalexperience associated with actual or potential tissue damage, ordescribed in terms of such damage. With the extension of longevity, theopportunities and duration of pain are raised. To estimate in theconservative way, the global anodyne consumption reaches around onehundred billion US dollars. Improving life quality through pain controlis an important subject. Among various pains, the factors of neuropathicpain are diverse, such as reduced distal circulation due to diabetesmellitus, neuron damage due to amputation or injury, viral infection andunknown reasons. Clinically, anodynes are divided into addictiveanodynes and non-addictive anodynes. The addictive anodyne mainlycomprises opiate, but the effect thereof to neuropathic pain is notsatisfactory. The non-addictive anodyne comprises a steroid type and anon-steroid type. The steroid anodyne relives pain mainly through ananti-inflammatory pathway. However, the steroid anodyne is nonspecific,and the side effects are significant. The long-term usage is prohibited.On the other hand, the non-steroid anodyne comprises a pain-relievingtype (such as Panadol) and an anti-inflammatory type (such as Aspirin).A non-steroid anti-inflammatory drug (NSAID) is now known to be safewith fewer side effects. The mechanism of a specific NSAID is throughinhibiting inducible nitric oxide synthase and cyclooxygenase-2 pathwaysto relieve pain (Turini and DuBois, Annual Rev Med 53:35, 2002; Handy etal, Br J Pharmacol 123:1119-1126, 1998; Osborne et al, Br J Pharmacol126:1840-1846, 1999). The product of NO or PGE2 catalyzed by induciblenitric oxide synthase or cyclooxygenase-2 is shown to be critical to theoccurrence, maintenance and sensitivity of pain in the central neuralsystem and periphery tissues (Moalem and Tracey, Brain Res Rev51:240-264, 2006). Compared to using nerve blockers for pain relieving,administering inducible nitric oxide synthase and cyclooxygenase-2inhibitors does not affect movement and neuron. Therefore, it is animportant aspect for drug development.

SUMMARY OF THE INVENTION

One object of the invention is to provide a novel sulfur-containingcompound. Said sulfur-containing compound can be chemically synthesizedand can significantly inhibit the functions of inflammatory proteins invitro. Furthermore, the sulfur-containing compound is shown to be ableto treat a disease associated with inducible nitric oxide synthaseand/or cyclooxygenase-2.

Another object of the invention is to provide a method for preparing thesulfur-containing compound mentioned above.

Still another object of the invention is to provide a method forinhibiting inducible nitric oxide synthase and/or cyclooxygenase-2.

Yet still another object of the invention is to provide a method fortreating a disease associated with inducible nitric oxide synthaseand/or cyclooxygenase-2 comprising administering a subject with saidsulfur-containing compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of the compound represented by formula 3 atdifferent concentrations on inducible nitric oxide synthase andcyclooxygenase-2 expressed by macrophages stimulated bylipopolysaccharide (LPS). A: results of Western blot; B: the statisticaldata of the effect of the compound represented by formula 3 at differentconcentrations on the expression of inducible nitric oxide synthase; C:the statistical data of the effect of the compound represented byformula 3 at different concentrations on the expression ofcyclooxygenase-2. Each test was repeated six times.

FIG. 2 shows the effect of the compound represented by formula 3 onneuropathic pain relief of chronic constriction injury in the sciaticnerve. A: results of the effect of the compound represented by formula 3administered intrathecally at different concentrations on paw withdrawallatency; B: maximum percent effect (MPE) of the compound represented byformula 3 administered intrathecally; C: the dose-response curve and 50%effective dose (ED₅₀) of the compound represented by formula 3calculated with MPE. Each point was repeated at least six times.

FIG. 3 shows the effect of the compound represented by formula 3 onneointimal proliferation of the carotid artery stimulated byballoon-induced angioplasty. A: control group; B: damage group; C:experiment group treated with the compound represented by formula 3; D,E, and F are the enlarged view of A, B, and C, respectively; G:statistical results of numeralizing the intima/media.

FIG. 4 shows the effect of the compound represented by formula 3 onmultiple sclerosis.

FIG. 5 shows the photographs of multiple sclerosis animal models withouttreatment (a) and treated with the compound represented by formula 3(b).

DETAILED DESCRIPTION OF THE INVENTION

The sulfur-containing compound according to the invention is representedby the following general formula 1,

-   -   wherein:    -   R¹ is selected from the group consisting of H, R⁵ and R⁵C(═O);    -   R² is selected from the group consisting of S and (O═)S(═O);    -   R³ is selected from the group consisting of H, CH₃ and        CH₂CH₂C(═O)OR⁵;    -   R⁴ is selected from the group consisting of R⁵, OR⁵,

N(R⁵)₂, NH₂, NHR⁵ and OH; and

-   -   R⁵ is selected from an alkyl group having one to six carbon        atoms and an unsubstituted or substituted phenyl group; and        preferably, R⁵ is selected from the group consisting of methyl,        ethyl, and unsubstituted phenyl,    -   provided that when R³ is CH₂CH₂C(═O)OR⁵, R⁴ is OR⁵; and    -   when R¹ is H, R¹ is S and R³ is H, R¹ is not CH₃.

According to the preferred embodiments of the invention, the compoundrepresented by general formula 1 is represented by one of the followingformulae 3 to 22,

The present invention also provides a method for preparing a compoundrepresented by the following general formula 2,

-   -   wherein:    -   R¹ is selected from the group consisting of H, R⁵ and R⁵C(═O);    -   R² is selected from the group consisting of S and (O═)S(═O);    -   R³ is selected from the group consisting of H, CH₃ and        CH₂CH₂C(═O)OR⁵;    -   R⁴ is selected from the group consisting of R⁵, OR⁵,

N(R⁵)₂, NH₂, NHR⁵ and OH; and

-   -   R⁵ is selected from an alkyl group having one to six carbon        atoms and an unsubstituted or substituted phenyl group;    -   provided that when R³ is CH₂CH₂C(═O)OR⁵, R⁴ is OR⁵, comprising:    -   (I) when R¹ is H and R² is S, reacting a compound represented by        the following general formula 23,

-   -   wherein R⁶ is selected from the group consisting of H and CH₃;    -   with 2-mercaptoethanol represented by the following formula 24,

-   -   to obtain a compound represented by the following general        formula 25

-   -   (II) when R¹ is R⁵C(═O), esterifying the compound represented by        general formula 25;    -   (III) when R¹ is R⁵, alkylating the compound represented by        general formula 25;    -   (IV) when R² is (O═)S(═O), oxidizing the compound represented by        general formula 25;    -   (V) when R¹ is R⁵C(═O) and R² is (O═)S(═O), esterifying and        oxidizing the compound represented by general formula 25;    -   (VI) when R¹ is R⁵ and R² is (O═)S(═O), alkylating and oxidizing        the compound represented by general formula 25; and    -   (VII) when R¹ is H, R² is S, R³ is H and R⁴ is CH₃, reacting a        compound represented by the following formula 27,

-   -   with 2-mercaptoethanol represented by formula 24 in the presence        of triethylamine.

Particularly, the method according to the invention is one of thefollowing methods:

(I) When R¹ is H and R² is S, the method of the invention comprisesreacting a compound represented by general formula 23

(wherein R⁶ is selected from the group consisting of H and CH₃; andpreferably is H)with 2-mercaptoethanol represented by formula 24 to obtain the compoundrepresented by general formula 25. Preferably, the above reaction iscarried out in the presence of triethylamine. In one embodiment of theinvention, the reactants are dissolved in acetone and reacted in icebath. In another aspect, when R³ is CH₂CH₂C(═O)OR⁵, the reaction isconducted in the absence of a solvent.

(II) When R¹ is R⁵C(═O), the method according to the invention comprisesesterifying the compound represented by general formula 25. Preferably,the above reaction is carried out in the presence of triethylamine. Inone embodiment of the invention, the reactants are dissolved indichloromethane. In one preferred embodiment of the invention, when R¹is CH₃C(═O), the method comprises reacting the compound represented bygeneral formula 25 with acetic anhydride. In one another preferredembodiment of the invention, when R¹ is C₆H₅C(═O), the method comprisesreacting the compound represented by general formula 25 with benzoylchloride.

(III) When R¹ is R⁵, the method according to the invention comprisesalkylating the compound represented by general formula 25. In onepreferred embodiment of the invention, when R¹ is CH₃, the methodcomprises reacting the compound represented by general formula 25 withCH₃I.

(IV) When R² is (O═)S(═O), the method according to the inventioncomprises oxidizing the compound represented by general formula 25.Preferably, the oxidation is carried out with hydrogen peroxide orm-chloroperoxybenzoic acid. In one embodiment of the invention, whenusing hydrogen peroxide, the oxidation is catalyzed by MnSO₄□H₂O, andthe reactants are dissolved in acetonitrile. When usingm-chloroperoxybenzoic acid to carry out the oxidation, the reactants aredissolved in dichloromethane.

(V) When R¹ is R⁵C(═O) and R² is (O═)S(═O), the method according to theinvention comprises esterifying and oxidizing the compound representedby general formula 25 as mentioned above.

(VI) When R¹ is R⁵ and R² is (O═)S(═O), the method according to theinvention comprises alkylating and oxidizing the compound represented bygeneral formula 25 as mentioned above.

(VII) When R¹ is H, R² is S, R³ is H and R⁴ is CH₃, the method accordingto the invention comprises reacting a compound represented by formula 27with 2-mercaptoethanol represented by formula 24 in the presence oftriethylamine.

The present invention also provides a method for inhibiting induciblenitric oxide synthase and/or cyclooxygenase-2.

The present invention further relates to a method for treating a diseaseassociated with inducible nitric oxide synthase and/or cyclooxygenase-2comprising administering a subject with the compound represented bygeneral formula 1. Because the compound represented by general formula 1has ability to inhibit the accumulation of inducible nitric oxidesynthase and/or cyclooxygenase-2, it is useful in treating the diseasesassociated with inducible nitric oxide synthase and/or cyclooxygenase-2.Many diseases have been reported to be related to the function ofinducible nitric oxide synthase and/or cyclooxygenase-2, such asarthritis (Cuzzocrea et al, Arthritis Rheum. 52:1929-40, 2005), multiplesclerosis (Misko et al, J Neuroimmunol. 61:195-204, 1995), inflammatorypain (Toriyabe et al, Anesthesiology 101, 983-990, 2004), and spinalcord injury (Lopez-Vales et al, Spine. 31:1100-6, 2006). Therefore, thedisease is preferably selected from the group consisting ofinflammation, atherosclerosis, neuropathic pain, inflammatory neointimalproliferation, arthritis, multiple sclerosis, inflammatory pain, andspinal cord injury. In one in vivo model of atherosclerosis in rat, thecompound represented by general formula 1 is shown to have the abilityto treat atherosclerosis. In another embodiment of the invention,administering the compound represented by general formula 1 throughintrathecal injection is effective in treating neuropathic pain.Furthermore, in the multiple sclerosis animal model, the compoundrepresented by general formula 1 is shown to have the ability to treatmultiple sclerosis.

The compound represented by general formula 1 can be administered orallyor through injection. Preferably, the compound is administered byinjection.

The following examples are given for the purpose of illustration onlyand are not intended to limit the scope of the present invention.

Preparation 1

2-Mercaptoethanol (1.80 g, 98%, 27.14 mmole) and triethylamine (0.53 mL,3.77 mmole) were added in a round bottom flask containing 22 mL ofacetone. Followed by stirring in a 0° C. ice bath, a solution of methylvinyl ketone (2.31 mL, 27.14 mmole) in 4 mL acetone was dropped into theflask slowly. After the addition, the temperature of the reaction wasraised to room temperature, and the reaction was continued for 16 hours.The solvent-free product was subject to silica gel column chromatographyto afford a thioether compound represented by the following formula 26(3.35 g, yield 100%),

Thioether compound represented by formula 26: colorless oil, IR (KBr)ν_(max) 3405, 1713, 1416, 1362, 1161, 1045, 1010 cm⁻¹; ¹H NMNR (CDCl₃,300 MHz) δ_(H) 3.73 (2H, dt, J=5.6, 5.4 Hz), 2.75 (4H, br s), 2.72 (2H,t, J=5.4 Hz), 2.46 (1H, t, J=5.6 Hz, OH) 2.17 (3H, s); ¹³C NMR (CDCl₃,75 MHz) δ_(C) 207.0 (qC), 60.6 (CH₂), 43.7 (CH₂), 35.6 (CH₂), 30.2(CH₃), 25.4 (CH₂); ESIMS m/z 171 [M+Na]⁺; HRESIMS m/z 171.0458 [M+Na]⁺(calculated for C₆H₁₂O₂SNa, 171.0456).

Preparation 2

The thioether compound represented by formula 26 (1.00 g, 6.76 mmole)prepared as Preparation 1 and MnSO₄□H₂O (23 mg, 0.14 mmole) were mixedwith acetonitrile (156 μL) in a 500 μL round bottom flask (Flask A) andthe mixture was stirred vigorously at room temperature. The aqueoussolutions of sodium hydrogen carbonate buffer (115 mL, 0.2 M, pH=8.0)and 30% hydrogen peroxide solution (3.38 μL) were charged into a 250 mLflask at 0° C. and stirred well, and then added slowly into Flask A.After reacting for 2 hours, ethyl acetate/isopropyl alcohol (3:1) wasadded for extraction (200 μL×6). The combined organic extract wasconcentrated under reduced pressure and the residue was purified over asilica gel column (eluted with hexane/EtOAc=1:3) for obtaining thecompound represented by formula 3 (1.03 g, yield 84%).

Compound represented by formula 3: colorless crystals, mp 59-60° C.; IR(KBr) ν_(max) 3416, 1715, 1416, 1366, 1311, 1120, 1009 cm⁻; ¹H NMR(CDCl₃, 300 MHz) δ_(H) 4.02 (2H, t, J=5.4 Hz), 3.36 (2H, t, J=7.3 Hz),3.21 (2H, t, J=5.4 Hz), 2.97 (2H, t, J=7.3 Hz), 2.20 (3H, s); ¹³C NMR(CDCl₃, 75 MHz) δ_(C) 204.9 (qC), 56.0 (CH₂), 55.7 (CH₂), 48.7 (CH₂),34.9 (CH₂), 29.7 (CH₃); ESIMS m/z 203 [M+Na]⁺; HRESIMS m/z 203.0354[M+Na]⁺ (calculated for C₆H₁₂O₄SNa, 203.0354).

Preparation 3

To a stirring solution of the thioether compound represented by formula26 (1.00 g, 6.76 mmole) prepared as Preparation 1 in dichloromethane (64mL) in a 125 mL round bottom flask was slowly addedm-chloroperoxybenzoic acid (2.92 g, 16.90 mmole) in batches. After theaddition, the reaction was monitored with TLC until the thioethercompound was completely consumed. Then, dichloromethane was removed, andthe resulted mixture was added with 100 mL of saturated sodium hydrogencarbonate solution and stirred vigorously. The resulted crude productwas extracted with ethyl acetate (100 mL×10) and the combined extractwas concentrated under reduced pressure. The obtained product waspurified with a silica gel column (eluted with hexane/EtOAc=1:3) toafford the compound represented by formula 3 (1.00 g, yield 82%).

Preparation 4

To a stirring solution of the compound represented by formula 3 (20.0mg, 0.114 mmole) and triethylamine (25 μL) in dichloromethane (2 mL) wasslowly added acetic anhydride (40 μL) at room temperature. The reactionwas continued for 16 hours and the solvent-free product was subjected tosilica gel column chromatography (eluted with hexane/EtOAc=2:3) forobtaining a compound represented by formula 4 (25.0 mg, yield 98%).

Compound represented by formula 4: colorless oil, IR (KBr) ν_(max) 1743,1720, 1366, 1317, 1234, 1125, 1070, 1036 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz)δ_(H) 4.52 (2H, t, J=5.8 Hz), 3.36 (2H, t, J=7.2 Hz), 3.34 (2H, t, J=5.8Hz), 3.03 (2H, t, J=7.2 Hz), 2.25 (3H, s), 2.11 (3H, s); ¹³C NMR (CDCl₃,75 MHz) δ_(C) 204.0 (qC), 170.2 (qC), 57.5 (CH₂), 52.8 (CH₂), 48.5(CH₂), 34.9 (CH₂), 29.8 (CH₃), 20.7 (CH₃); ESIMS m/z 245 [M+Na]⁺;HRESIMS m/z 245.0458 [M+Na]⁺ (calcd for C₈H₁₄O₅SNa, 245.0460).

Preparation 5

To a stirring solution of the compound represented by formula 3 (20.0mg, 0.114 mmole) and triethylamine (25 μL) in dichloromethane (2 mL) wasslowly added benzoyl chloride (50 μL) at room temperature. The reactionwas continued for 5 hours and the solvent-free product was subjected tosilica gel column chromatography (eluted with hexane/EtOAc=5:3) forobtaining a compound represented by formula 5 (29.8 mg, yield 92%).

Compound represented by formula 5: colorless crystals, mp=79-80° C.; IR(KBr) ν_(max) 1714, 1710, 1315, 1276, 1133, 1119 cm⁻¹; ¹H NMR (CDCl₃,300 MHz) δ_(H) 8.04 (2H, d, J=7.5 Hz), 7.60 (1H, t, J=7.5 Hz), 7.47 (2H,t, J=7.5 Hz), 4.78 (2H, t, J=5.7 Hz), 3.48 (2H, t, J=5.7 Hz), 3.41 (2H,t, J=7.2 Hz), 3.03 (2H, t, J=7.2 Hz), 2.21 (3H, s); ¹³C NMR (CDCl₃, 75MHz) δ_(C) 203.8 (qC), 165.8 (qC), 133.5 (CH), 129.6 (CH×2), 129.0 (qC),128.6 (CH×2), 58.0 (CH₂), 53.0 (CH₂), 48.6 (CH₂), 34.9 (CH₂), 29.7(CH₃); ESIMS m/z 307 [M+Na]⁺; HRESIMS m/z 307.0613 [M+Na]⁺ (calcd forC₁₃H₁₆O₅SNa, 307.0616).

Preparation 6

Similar to Preparation 1, α,β-unsaturated carbonly compounds includingethyl vinyl ketone, ethyl acrylate, N,N-dimethylacrylamide,4-acryloylmorpholine, and acrylamide were independently reacted with2-mercaptoethanol to give the corresponding thioether compoundsrepresented by formulas 6 (yield 100%), 8 (yield 100%), 10 (yield 100%),12 (yield 100%), and 14 (yield 90%), respectively. The above reactionswere carried out in the presence of acetone, except for that ofpreparing the compound represented by formula 14 required the presenceof a more polar solvent (methanol/acetone=1:1), due to theunsatisfactory solubility of acrylamide. The intermediates with formulas6, 8, 10, 12, and 14 were oxidized with hydrogen peroxide, similar toPreparation 2 for obtaining compounds represented by formula 7 (yield87%), 9 (yield 85%), 11 (yield 87%), 13 (yield 83%), and 15 (yield 84%),respectively. Furthermore, the reaction of ethyl acrylate and2-mercaptoethanol, proceeded without the presence of a solvent, affordedboth compounds represented by formula 8 (yield 45%) and formula 16(yield 22%). Compound represented by formula 17 was prepared byoxidizing the compound represented by formula 16 with hydrogen peroxide(yield 84%).

Compound represented by formula 6: colorless oil; IR (KBr) ν_(max) 3418,1714, 1458, 1411, 1375, 1361, 1113, 1046, 1013 cm⁻¹; ¹H NMR (CDCl₃, 300MHz) δ_(H) 3.75 (2H, t, J=6.2 Hz), 2.70-2.80 (6H, m), 2.48 (2H, q, J=7.3Hz), 1.06 (3H, t, J=7.3 Hz); ¹³C NMR (CDCl₃, 75 MHz) δ_(C) 209.9 (qC),60.6 (CH₂), 42.0 (CH₂), 35.9 (CH₂), 34.8 (CH₂), 25.3 (CH₂), 7.3 (CH₃);ESIMS m/z 185 [M+Na]⁺; HRESIMS m/z 185.0611 [M+Na]⁺ (calcd forC₇H₁₄O₂SNa, 185.0612).

Compound represented by formula 7: colorless oil; mp=44-45° C.; IR (KBr)ν_(max) 3419, 1715, 1312, 1280, 1123 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ_(H)4.01 (2H, t, J=5.2 Hz), 3.36 (2H, t, J=7.3 Hz), 3.19 (2H, t, J=5.2 Hz),2.93 (2H, t, J=7.3 Hz), 2.47 (2H, q, J=7.3 Hz), 1.01 (3H, t, J=7.3 Hz);¹³C NMR (CDCl₃, 75 MHz) δ_(C) 207.6 (qC), 56.0 (CH₂), 55.7 (CH₂), 48.8(CH₂), 35.8 (CH₂), 33.6 (CH₂), 7.5 (CH₃); ESIMS m/z 217 [M+Na]⁺; HRESIMSm/z 217.0508 [M+Na]⁺ (calcd for C₇H₁₄O₄SNa, 217.0510).

Compound represented by formula 8: colorless oil; IR (KBr) ν_(max) 3440,1732, 1373, 1249, 1185, 1044 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ_(H) 4.16(2H, q, J=7.1 Hz), 3.74 (2H, t, J=6.1 Hz), 2.82 (2H, t, J=7.1 Hz), 2.74(2H, t, J=6.1 Hz), 2.62 (2H, t, J=7.1 Hz), 1.27 (3H, t, J=7.1 Hz); ¹³CNMR (CDCl₃, 75 MHz) δ_(C) 172.1 (qC), 60.8 (CH₂×2), 35.1 (CH₂), 34.9(CH₂), 26.8 (CH₂), 14.2 (CH₃); ESIMS m/z 201 [M+Na]⁺; HRESIMS m/z201.0563 [M+Na]⁺ (calcd for C₇H₁₄O₃SNa, 201.0561).

Compound represented by formula 9: colorless oil; IR (KBr) ν_(max) 3503,1732, 1313, 1281, 1120, 1065 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ_(H) 4.19(2H, q, J=7.1 Hz), 4.12 (2H, t, J=5.0 Hz), 3.46 (2H, t, J=7.4 Hz), 3.25(2H, t, J=5.0 Hz), 2.88 (2H, t, J=7.4 Hz), 1.28 (3H, t, J=7.1 Hz); ¹³CNMR (CDCl₃, 75 MHz) δ_(C) 170.7 (qC), 61.5 (CH₂), 56.2 (CH₂), 55.6(CH₂), 49.9 (CH₂), 26.8 (CH₂), 14.0 (CH₃); ESIMS m/z 233 [M+Na]⁺;HRESIMS m/z 233.0458 [M+Na]⁺ (calcd for C₇H₁₄O₅SNa, 233.0460).

Compound represented by formula 10: colorless oil; IR (KBr) ν_(max)3399, 1630, 1500, 1403, 1143, 1048, 1014 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz)δ_(H) 3.77 (2H, t, J=5.9 Hz), 3.02 (3H, s), 2.96 (3H, s), 2.87 (2H, t,J=7.2 Hz), 2.75 (2H, t, J=5.9 Hz), 2.62 (2H, t, J=7.2 Hz); ¹³C NMR(CDCl₃, 75 MHz) δ_(C) 171.1 (qC), 60.8 (CH₂), 37.0 (CH₃), 35.4 (CH₃×1,CH₂×1), 33.5 (CH₂), 26.8 (CH₂); ESIMS m/z 200 [M+Na]⁺; HRESIMS m/z200.0719 [M+Na]⁺ (calcd for C₇H₁₅NO₂SNa, 200.0721).

Compound represented by formula 11: colorless oil; IR (KBr) ν_(max)3371, 1634, 1503, 1405, 1312, 1278, 1119, 1065 cm⁻¹; ¹H NMR (CDCl₃, 300MHz) δ_(H) 4.06 (2H, t, J=5.2 Hz), 3.49 (2H, t, J=7.2 Hz), 3.25 (2H, t,J=5.2 Hz), 3.03 (3H, s), 2.94 (3H, s), 2.87 (2H, t, J=7.2 Hz); ¹³C NMR(CDCl₃, 75 MHz) δ_(C) 169.4 (qC), 56.2 (CH₂), 56.0 (CH₂), 50.3 (CH₂),37.1 (CH₃), 35.7 (CH₃), 25.7 (CH₂); ESIMS m/z 232 [M+Na]⁺; HRESIMS m/z232.0618 [M+Na]⁺ (calcd for C₇H₁₅NO₄SNa, 232.0619).

Compound represented by formula 12: colorless oil; IR (KBr) ν_(max)3418, 1633, 1463, 1437, 1271, 1248, 1115, 1067, 1028 cm⁻¹; ¹H NMR(CDCl₃, 300 MHz) δ_(H) 3.76 (2H, t, J=6.0 Hz), 3.69 (4H, m), 3.63 (2H,m), 3.48 (2H, dd, J=4.5, 4.9 Hz), 2.87 (2H, t, J=7.2 Hz), 2.74 (2H, t,J=6.0 Hz), 2.63 (2H, t, J=7.2 Hz); ¹³C NMR (CDCl₃, 75 MHz) δ_(C) 169.8(qC), 66.6 (CH₂), 66.3 (CH₂), 60.8 (CH₂), 45.7 (CH₂), 41.9 (CH₂), 35.2(CH₂), 33.2 (CH₂) 26.8 (CH₂); ESIMS m/z 242 [M+Na]⁺; HRESIMS m/z242.0815 [M+Na]⁺ (calcd for C₉H₁₇NO₃SNa, 242.0813).

Compound represented by formula 13: colorless needles; mp=109-110° C.;IR (KBr) ν_(max) 3420, 1637, 1452, 1311, 1275, 1117, 1067, 1028 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz) δ_(H) 4.06 (2H, t, J=5.2 Hz), 3.57-3.67 (6H, m),3.49 (2H, t, J=7.2 Hz), 3.47 (2H, m), 3.25 (2H, t, J=5.2 Hz), 2.86 (2H,t, J=7.2 Hz); ¹³C NMR (CDCl₃, 75 MHz) δ_(C) 168.0 (qC), 66.5 (CH₂), 66.3(CH₂), 56.2 (CH₂), 56.0 (CH₂), 50.2 (CH₂), 45.7 (CH₂), 42.3 (CH₂) 25.3(CH₂); ESIMS m/z 274 [M+Na]⁺; HRESIMS m/z 274.0722 [M+Na]⁺ (calcd forC₉H₁₇NO₅SNa, 274.0725).

Compound represented by formula 14: white powder; IR (KBr) ν_(max) 3354,3196, 1661, 1414 cm⁻¹; ¹H NMR (pyridine-d5, 300 MHz) δ_(H) 4.02 (2H, t,J=6.7 Hz), 3.13 (2H, t, J=7.2 Hz), 2.93 (2H, t, J=6.7 Hz), 2.82 (2H, t,J=7.2 Hz); ¹³C NMR (pyridine-d5, 75 MHz) δ_(C) 174.3 (qC), 61.7 (CH₂),36.6 (CH₂), 35.2 (CH₂), 28.1 (CH₂); ESIMS m/z 172 [M+Na]⁺; HRESIMS m/z172.0407 [M+Na]⁺ (calcd for C₅H₁₁NO₂SNa, 172.0408).

Compound represented by formula 15: white powder; IR (KBr) ν_(max) 3370,3200, 1661, 1395 cm⁻¹; ¹H NMR (pyridine-d5, 300 MHz) δ_(H) 4.35 (2H, t,J=5.6 Hz), 4.06 (2H, t, J=7.6 Hz), 3.64 (2H, t, J=5.6 Hz), 3.25 (2H, t,J=7.6 Hz); ¹³C NMR (pyridine-d5, 75 MHz) δ_(C) 174.3 (qC), 61.7 (CH₂),36.6 (CH₂), 35.2 (CH₂), 28.1 (CH₂); ESIMS m/z 204 [M+Na]⁺; HRESIMS m/z204.0305 [M+Na]⁺ (calcd for C₅H₁₁NO₄SNa, 204.0306).

Compound represented by formula 16: colorless oil; IR (KBr) ν_(max)3438, 1731, 1377, 1299, 1206, 1162, 1043 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz)δ_(H) 4.10 (2H, q, J=7.0 Hz), 4.05 (2H, q, J=7.0 Hz), 3.65 (2H, t, J=6.0Hz), 2.54-2.74 (5H, m), 2.28 (2H, m), 1.91 (2H, m), 1.20 (3H, t, J=7.0Hz), 1.18 (3H, t, J=7.0 Hz); ¹³C NMR (CDCl₃, 75 MHz) δ_(C) 174.1 (qC),172.8 (qC), 60.89 (CH₂), 60.86 (CH₂), 60.6 (CH₂), 45.3 (CH), 35.5 (CH₂),33.6 (CH₂), 31.7 (CH₂) 26.7 (CH₂), 14.22 (CH₃), 14.18 (CH₃); ESIMS m/z301 [M+Na]⁺; HRESIMS m/z 301.1084 [M+Na]⁺ (calcd for C₁₂H₂₂O₅SNa,301.1086).

Compound represented by formula 17: colorless oil; IR (KBr) ν_(max)3439, 1732, 1380, 1312, 1290, 1206, 1120 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz)δ_(H) 4.21 (2H, q, J=7.1 Hz), 4.15 (2H, q, J=7.1 Hz), 4.11 (2H, t, J=5.3Hz), 3.72 (1H, dd, J=14.2, 9.3 Hz), 3.25 (2H, t, J=5.3 Hz), 3.21 (1H,dd, J=14.2, 4.9 Hz), 3.13 (1H, m), 2.39 (2H, t, J=7.4 Hz), 2.04 (2H, t,J=7.4 Hz), 1.30 (3H, t, J=7.1 Hz), 1.27 (3H, t, J=7.1 Hz); ¹³C NMR(CDCl₃, 75 MHz) δ_(C) 173.0 (qC), 172.4 (qC), 61.6 (CH₂), 60.8 (CH₂),56.4 (CH₂), 55.1 (CH₂), 55.8 (CH₂), 38.7 (CH), 31.1 (CH₂) 27.2 (CH₂),14.1 (CH₃), 14.0 (CH₃); ESIMS m/z 333 [M+Na]⁺; HRESIMS m/z 333.0986[M+Na]⁺ (calcd for C₁₂H₂₂O₇SNa, 333.0987).

Anti-Inflammation Assay In Vitro

A mouse macrophage cell line, RAW 264.7, purchased from the AmericanType Culture Collection (ATCC□NO TIB-71) was chosen in the in vitromodel. The cells were cultured in DMEM (Dulcbecco/s Modified Eaglemedium) containing 10% fetal bovine serum (FBS) and penicillin G (100U/ml) and streptomycin (100 μg/ml) at 37° C. and 5% CO₂. When reaching80% confluence, the cells were subcultured with trypsin. The cells weresubjected to an anti-inflammation assay after subcultured for 36 hours.3×10⁶ RAW264.7 cells were cultured in a 10-cm culture dish andadministered with lipopolysaccharide (LPS, 0.01 μg/ml; Sigma L2654).After 16 hours, the cells were collected. In an experiment group, thecompound represented by formula 3 was added into the culture dish andfollowed by LPS before 10 minutes.

Assay for Protein Expression of Inducible Nitric Oxide Synthase and/orCyclooxygenase-2

The collected RAW264.7 cells were dissolved with 200 μL of 4° C. lysisbuffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% TritonX-100, 0.1 mM EDTA,0.1 mM EGTA, 10 μg PMSF, 1 μg/mL aprotinin, 20 mM NaF, and 0.2 mMNa₃VO₄). The samples were centrifuged at 25,000 g for 30 minutes at 4°C. for removing the pellet. The supernatant was assayed with Bio-Rad DCprotein assay kit (Bio-Rad Laboratories, Hercules, Calif., USA) and theabsorbance was read with an ELISA reader (Thermo Electron Corporation,USA) for estimating the protein contents. The calibrated samples withequal volumes were added with a sample buffer (2% SDS, 10% glycerol,0.1% bromophenol blue, 10% 2-mercaptoethanol, and 50 mM Tris) of thesame volume. Proteins were separated with 10% SDS-PAGE and transferredto a PVDF membrane (0.45 mm, Immobilon-P, Millipore, Bedford, Mass.,USA) (1.5 A, 4° C., 2.5 hours). The transferred PVDF membranes wereblocked with TTBS (Tris-HCl 20 mM, NaCl 137 mM, pH 7.4 and 0.1% Tween20) containing 5% skim milk at room temperature for 1 hour and reactedwith polyclonal anti-inducible nitric oxide synthase antibody(Transduction Laboratories, Lexington, Ky., USA) or polyclonalanti-cyclooxygenase-2 antibody (Cayman, Ann Arbor, Mich., USA) at roomtemperature for 3 hours. After washed with TTBS three times, the sampleswere reacted with HRP-conjugated anti-rabbit IgG antibody (1:2000) atroom temperature for 1 hour. After washed with TTBS for three times, anenhanced chemiluminescence detection kit was used for reating with thePVDF membrane and exposed with an X-ray film (Kodak X-OMAT LS, Kodak,Rochester, N.Y., USA) for detecting the protein expression. The relativeamount was calculated with Image-Pro plus 4.5 software (MediaCybernetics, Silver Spring, USA). The group added with only LPS wastaken as 100%. β-actin (monoclonal antibody, Sigma, St Louis, Mo., USA)was taken as an internal control.

The results are shown in FIG. 1. 10 to 30 μM of the compound representedby formula 3 significantly inhibits the effect of inducible nitric oxidesynthase of macrophages stimulated by LPS (0.01 μg/ml) and 50%inhibition concentration (IC₅₀) is 3.64±0.28 μM. 5 to 30 μM of thecompound represented by formula 3 also significantly inhibits the effectof cyclooxygenase-2 of macrophages stimulated by LPS (0.01 μg/ml) andthe IC₅₀ is 32.1±8.1 μM. However, at a concentration of 50 μM thecompound represented by formula 3 inhibits the protein expression ofβ-actin.

Animal Model Intrathecal Catheter Operation

The operation was performed according to the description of Wen et al,Brain Res 963:1-7, 2003. The dura mater of male Wistar rats was openednear the foramen magnum and planted with an 8.5-cm PE tube (outerdiameter: 0.014 inches, inner diameter: 0.008 inches). The drugs workednear the lumbar, and the end of injection was fixed on the head. Animalsthose had deficient in motor or blood in the intrathecal catheter wereabandoned.

Animal Model of Neuropathic Pain

The model was established similar to the sciatic nerve chronicconstriction injury (CCI) established by Bennett and Xie (Pain33:87-107, 1988). 4-O cat cut line was used to tie four nodes on thesciatic nerve. After one week, the behavior of thermal hyperalgesia wasstimulated. The behavior of thermal hyperalgesia was assayed similar tothe method established by Hargreaves et al (Pain 32:77-88, 1988) andrecorded with an analgesiometer (IITC Inc., Woodland Hills, Calif., USA)for assessing the effect of the compound represented by formula 3 onneuropathic pain relief.

The data of each animal were exhibited with the maximum percent effect(MPE) and analyzed statistically.

MPE (%)=(latency after drug administered−basal latency)/(30seconds−basal latency)×100%

If a higher MPE was obtained, the effect of pain relief was better, andthe maximum value of MPE was 100.

The results of neuropathic pain relief are shown in FIG. 2. Thermalhyperalgesia started on day 7 after sciatic nerve injury. The pawwithdrawal latency decreased from 30 seconds to about 14 seconds.Intrathecal injections of the compound represented by formula 3 of 0.1,0.5, 1, or 5 μg all show significant effect on thermal hyperalgesiainhibition. After calculating the MPE, the 50% effective dose (ED₅₀) is0.75±0.05 μg.

Atherosclerosis Model in Rat

The operation was performed according to the references of Berger et al(Atherosclerosis 175:229-234, 2004) and Chen et al (Naunyn SchmiedebergsArch Pharmacol 368:127-133, 2003). The animals were anesthetized with2.5% isoflurane (mixed air and oxygen of 1:1) with a 2F probe ballooncatheter. The catheter was pre-filled with saline solution andpositioned from the external carotid artery of the right neck into theright common carotid artery. When the catheter entered about 1.5 cm ofthe right common carotid artery, the balloon was inflated and rubbedforward and backward three times. Then, the catheter was removed and theexternal carotid artery was ligated and the incision was sutured. Theanimals were sacrificed after three weeks and the common carotid arteryfrom both sides were taken and fixed with 4% paraformaldehyde for oneday and sliced (3 μm) and subjected to H & E stain. The samples wereobserved under an optical microscopy and photoed. The thickness ofneointimal proliferation was determined. The right side of each rat wastaken as the experiment group and the left side was as the controlgroup.

In the experiment group, the compound represented by formula 3 wasadministered. The preliminary result shows that neointimal proliferationis significantly improved by administering the compound once a day.

The effect of anti-atherosclerosis on the atherosclerosis stimulated byballoon-induced angioplasty is shown in FIG. 3. On day 24 afteroperation, neointimal proliferation in carotid artery was observed inthe slice. The compound represented by formula 3 was administered fromday 10 to 24 after operation through hypodermic injection with 3 mg/kg aday, and it improves the neointimal proliferation stimulated byballoon-induced angioplasty. When numeralizing the intima/media, it isfound that the compound represented by formula 3 improvesatherosclerosis significantly.

Multiple Sclerosis Animal Model

Female Lewis rats weighted between 280 to 300 g were used in themultiple sclerosis (MS, also called Encephalomyelitis; EAE) model(Boulerne et al, J Neurosci. 2002; 22:123-32, 2004). The animals wereanesthetized with 2.5% isoflurane, and 50 μL of Complete Freund'sAdjuvant (Sigma) containing 1 mg inactivated Mycobacterium tuberculosisH37Ra and 50 μg guinea pig myelin basic protein (MBP) was subcutaneousinjected from the posterior paw for 100% immunizing. In the controlgroup, the rats were injected with Freund's complete adjuvant containinginactivated Mycobacterium tuberculosis H37Ra and without guinea pigmyelin basic protein. The ill was observed on day 11 to 12 afterimmunization, and reached the peak on day 14 to 15.

The neural function was assessed on day 15. The scores represents: 0: noclinical symptom; 1: function loss in the tail; 2: partial paralysis inthe posterior limbs; 3: complete paralysis in the posterior limbs; 4:paralysis in the four limbs; 5: complete paralysis in the whole body; 6:death (Liu et al. J Neuroimmunol. 2003; 139:27-35, 1998).

The rats were divided into four groups, and each group containing fouranimals:

-   -   Control group: Rats were subcutaneous injected from the        posterior paw with Freund's complete adjuvant containing        inactivated Mycobacterium tuberculosis H37Ra and without guinea        pig myelin basic protein. 20 μL of normal saline was        administered through the intrathecal catheter twice a day for        five days from day 7.    -   Multiple sclerosis group: Rats were subcutaneous injected from        the posterior paw with Freund's complete adjuvant containing        inactivated Mycobacterium tuberculosis H37Ra and guinea pig        myelin basic protein. 20 μL of normal saline was administered        through the intrathecal catheter twice a day for five days from        day 7.    -   Multiple sclerosis with treatment group: Rats were subcutaneous        injected from the posterior paw with Freund's complete adjuvant        containing inactivated Mycobacterium tuberculosis H37Ra and        guinea pig myelin basic protein. 10 μg/20 μL of compound        represented by formula 3 was administered through the        intrathecal catheter twice a day for five days from day 7.    -   Normal rats with treatment group: 10 μg/20 μL of compound        represented by formula 3 was administered through the        intrathecal catheter twice a day for five days from day 7.

The scores of the control group and normal rats with treatment group are0. The score of multiple sclerosis group is 4.6±0.2 and that of themultiple sclerosis with treatment group is 0.5±0.3. It shows thatadministering the compound represented by formula 3 through theintrathecal catheter significantly represses the progress of multiplesclerosis (FIGS. 4 and 5).

Statistics

All data were exhibited with average ±standard deviation S.E.M. andanalyzed with one-way analysis of variance (ANOVA) and Dunnett's test.P<0.05 shows significant difference. In the anti-inflammation assay invitro, administering only LPS was taken as 100%. In the animal model,each group was repeated four to eight times.

While embodiments of the present invention have been illustrated anddescribed, various modifications and improvements can be made by personsskilled in the art. It is intended that the present invention is notlimited to the particular forms as illustrated, and that all themodifications not departing from the spirit and scope of the presentinvention are within the scope as defined in the following claims.

1. A compound represented by the following general formula 1,

wherein: R¹ is selected from the group consisting of H, R⁵ and R⁵C(═O); R² is selected from the group consisting of S and (O═)S(═O); R³ is selected from the group consisting of H, CH₃ and CH₂CH₂C(═O)OR⁵; R⁴ is selected from the group consisting of R⁵, OR⁵,

N(R⁵)₂, NH₂, NHR⁵ and OH; and R⁵ is selected from an alkyl group having one to six carbon atoms and an unsubstituted or substituted phenyl group; provided that when R³ is CH₂CH₂C(═O)OR⁵, R⁴ is OR⁵; and when R¹ is H, R² is S and R³ is H, R⁴ is not CH₃.
 2. The compound according to claim 1, wherein R⁵ is selected from the group consisting of methyl, ethyl, and unsubstituted phenyl.
 3. The compound according to claim 1, which is selected from the group consisting of the compounds represented by the following formulae 3 to 22;


4. A method for preparing a compound represented by the following general formula 2,

wherein: R¹ is selected from the group consisting of H, R⁵ and R⁵C(═O); R² is selected from the group consisting of S and (O═)S(═O); R³ is selected from the group consisting of H, CH₃ and CH₂CH₂C(═O)OR⁵; R⁴ is selected from the group consisting of R⁵, OR⁵,

N(R⁵)₂, NH₂, NHR⁵ and OH; and R⁵ is selected from an alkyl group having one to six carbon atoms and an unsubstituted or substituted phenyl group; provided that when R³ is CH₂CH₂C(═O)OR⁵, R⁴ is OR⁵; comprising: (I) when R¹ is H and R² is S, reacting a compound represented by the following general formula 23,

wherein R⁶ is selected from the group consisting of H and CH₃; with 2-mercaptoethanol represented by the following general formula 24,

to obtain a compound represented by the following general formula 25,

(II) when R¹ is R⁵C(═O), esterifying the compound represented by general formula 25; (III) when R¹ is R⁵, alkylating the compound represented by general formula 25; (IV) when R² is (O═)S(═O), oxidizing the compound represented by general formula 25; (V) when R¹ is R⁵C(═O) and R² is (O═)S(═O), esterifying and oxidizing the compound represented by general formula 25; (VI) when R¹ is R⁵ and R² is (O═)S(═O), alkylating and oxidizing the compound represented by general formula 25; and (VII) when R¹ is H, R² is S, R³ is H and R⁴ is CH₃, reacting a compound represented by the following formula 27

with 2-mercaptoethanol represented by formula 24 in the presence of triethylamine.
 5. The method according to claim 4, wherein R⁶ is H.
 6. The method according to claim 4, wherein the compounds represented by formulae 23 and 24 are reacted in the presence of triethylamine.
 7. The method according to claim 4, wherein the compounds represented by formulae 23 and 24 are dissolved in acetone.
 8. The method according to claim 4, wherein when R³ is CH₂CH₂C(═O)OR⁵, the compounds represented by formulae 23 and 24 are reacted in the absence of a solvent.
 9. The method according to claim 4, wherein the esterification is carried out in the presence of triethylamine.
 10. The method according to claim 4, wherein reactants of the esterification are dissolved in dichloromethane.
 11. The method according to claim 4, wherein when R¹ is CH₃C(═O), the esterification comprises reacting the compound represented by general formula 25 with acetic anhydride.
 12. The method according to claim 4, wherein when R¹ is C₆H₅C(═O), the esterification comprises reacting the compound represented by general formula 25 with benzoyl chloride.
 13. The method according to claim 4, wherein when R¹ is CH₃, the alkylation comprises reacting the compound represented by general formula 25 with CH₃I.
 14. The method according to claim 4, wherein the oxidation is carried out with hydrogen peroxide.
 15. The method according to claim 14, wherein the oxidation is catalyzed by MnSO₄□H₂O.
 16. The method according to claim 14, wherein reactants of the oxidation are dissolved in acetonitrile.
 17. The method according to claim 4, wherein the oxidation is carried out with m-chloroperoxybenzoic acid.
 18. The method according to claim 17, wherein reactants of the oxidation are dissolved in dichloromethane.
 19. A method for inhibiting inducible nitric oxide synthase and/or cyclooxygenase-2 comprising administering a subject with the compound according to claim
 1. 20. The method according to claim 19, wherein the compound is represented by the following formula 3


21. A method for treating a disease associated with inducible nitric oxide synthase and/or cyclooxygenase-2 comprising administering a subject with the compound according to claim
 1. 22. The method according to claim 21, wherein the disease is selected from the group consisting of inflammation, atherosclerosis, neuropathic pain, inflammatory neointimal proliferation, arthritis, multiple sclerosis, inflammatory pain, and spinal cord injury.
 23. The method according to claim 21, wherein the compound is administered by injection.
 24. The method according to claim 21, wherein the compound is represented by the following formula 3, 